Stufies on the Biochemical and Genetic Bases of the Multiplicity of Cytochrome P-450

细胞色素P-450多样性的生化和遗传基础研究

• 批准号: 58060002
• 负责人: SATO Ryo
• 金额: $ 133.44万
• 依托单位: Institute for Protein Research, Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Specially Promoted Research
• 财政年份: 1983
• 资助国家: 日本
• 起止时间: 1983 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-58060002/
• 关键词: Cytochrome P-450; Molecular Multiplicity; cDNA; Regulation of Gene Expression; Enzyme Induction; 酵素誘導; 遺伝子; ヌクレオチド配列; アミノ酸配列

Multiple forms of cytochrome P-450 occur in liver microsomes and administration of various drugs to animals leads to the induction of a specific form or forms of cytochrome P-450. This project was undertaken to elucidate the biochemical and genetic bases of this multiplicity of mammalian cytochrome P-450. The major results obtained in this project may be summarized as follows. First, 15 forms of cytochrome P-450 were purified from liver microsomes of untreated and variously drug-treated rabbits and their properties were examined in detail. Secondly, 16 cDNA clones including 8 full-length ones coding for different forms of rat and rabbit liver microsomal cytochrome P-450 were isolated and their nucleotide sequences determined. Comparison of their deduced primary structures and those determined in other laboratories leads to the conclusion that the mammalian P-450 gene superfamily consists of at least 5 families and some of the families are further classified into several subfamilies. Furthermore, microheterogeneity can be detected in the major phenobarbital-inducible form in rabbit liver. Thirdly, 4 rat P-450 genes, 1 rabbit P-450 gene and 2 human adrenal cortex P-450 genes were cloned and their structures were determined. In the rat P-450c gene three elements regulating its expression were detected in its 5' upstream region and one of them was identified as an enhancer involved in drug-mediated expression. Finally, Rat P-450d and a rabbit P-450 were successfully expressed in yeast cells. The P-450 proteins thus isolated from yeast cells were found to show relatively broad substrate specificities in spite of the fact that these P-450 proteins are homogeneous with respect to primary structure.

多种形式的细胞色素P-450存在于肝微粒体中，给动物施用各种药物可诱导一种或多种特定形式的细胞色素P-450。本项目旨在阐明哺乳动物细胞色素P-450多样性的生化和遗传基础。本项目取得的主要成果可以总结如下。首先，从未经处理和不同药物处理的家兔的肝微粒体中纯化了15种细胞色素P-450，并详细检查了它们的性质。其次，分离了16个cDNA克隆，其中8个全长克隆编码不同形式的大鼠和家兔肝微粒体细胞色素P-450，并测定了它们的核苷酸序列。将其推断的初级结构与其他实验室测定的结构进行比较，得出哺乳动物P-450基因超家族至少由5个家族组成，其中一些家族进一步分为几个亚家族。此外，在兔肝脏中，苯巴比妥诱导的主要形式存在微异质性。再次，克隆了4个大鼠P-450基因、1个家兔P-450基因和2个人肾上腺皮质P-450基因，并确定了它们的结构。在大鼠P-450c基因的5′上游区检测到3个调控其表达的元件，其中一个被鉴定为参与药物介导表达的增强子。最后，在酵母细胞中成功表达了大鼠P-450d和家兔P-450。从酵母细胞中分离出的P-450蛋白显示出相对广泛的底物特异性，尽管这些P-450蛋白在初级结构方面是均匀的。

项目成果

K.Sogawa;O.Gotoh;K.Kawajiri;Y.Fujii-Kuriyama: Proc.Natl.Acad.Sci.USA. 81. 5066-5070 (1984)

K.Sokawa；O.Gotoh；K.Kawajiri；Y.Fujii-Kuriyama：Proc.Natl.Acad.Sci.USA。

Y.Aoyama;Y.Yoshida;R.Sato: J.Biol.Chem.259. 1661-1666 (1984)

Y.Aoyama；Y.Yoshida；R.Sato：J.Biol.Chem.259。

M.SaKaguchi;K.Mihara;R.Sato: Proc.Natl.Acad.Sci.USA. 81. 3361-3364 (1984)

M.SaKaguchi；K.Mihara；R.Sato：Proc.Natl.Acad.Sci.USA。

K.Kawajiri, K.Sogawa, O.Gotoh, Y.Tagashira, M.Muramatsu & Y.Fujii-Kuriyama: "Molecular cloning of a complementary DNA to 3-methylcholanthrene-inducible cytochrome P-450 mRNA from rat liver." Journal of Biochemistry. 94. 1465-1473 (1983)

K.Kawajiri、K.Sokawa、O.Gotoh、Y.Tagashira、M.Muramatsu

Y.Higashi, H.Yoshioka, M.Yamane, O.Gotoh & Y.Fujii-Kuriyama: "Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: A pseudogene and a genuine gene." Proceedings of the National Academy of Sciences, USA. 8

Y.Higashi、H.Yoshioka、M.Yamane、O.Gotoh